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J S Fink

Publications and source records attributed to J S Fink.

At least 37 records · Page 2Linked to original sources
PubMed1996

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Animals↗
PubMed1996

Characterization and expression of the human A2a adenosine receptor gene.

The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans approximately 9-10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.

Amino Acid Sequence↗
PubMed1995

LIF-mediated activation of STAT proteins after neuronal injury in vivo.

Leukemia inhibitory factor (LIF) is a member of a family of neuropoietic cytokines which influence neuronal survival, differentiation and response to injury. In cell lines LIF activates the Jak/Tyk tyrosine kinases and the STAT family of transcription factors. We have investigated whether the Jak-STAT intracellular signaling pathway is activated by LIF after neuronal injury in vivo. Axotomy of postganglionic sympathetic nerves resulted in sustained activation of members of the STAT transcription factor family. This activation is dependent on LIF as axotomy failed to activate STAT proteins in LIF-deficient mice. These data indicate that LIF-dependent activation of STAT proteins is one of the signal transduction pathways activated after neuronal injury.

Animals↗
PubMed1995

C/EBP-related sites in addition to a STAT site are necessary for ciliary neurotrophic factor-leukemia inhibitory factor-dependent transcriptional activation by the vasoactive intestinal peptide cytokine response element.

The neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) regulate VIP gene expression through a cytokine response element (CyRE) which interacts with members of the STAT transcription factor family. The CyRE STAT site is, however, insufficient to mediate full transcriptional activation by CNTF/LIF, suggesting that other sequences and nuclear proteins are also important. As C/EBP proteins participate in the transcriptional effects of the related cytokine, interleukin-6, we investigated the role of possible C/EBP-binding sites in the response of the VIP CyRE to CNTF/LIF. Using DNase I footprinting, transactivation studies, DNA mobility shift assays, and mutational analysis, three sites within the VIP CyRE were identified as C/EBP-related binding sites and shown to be important to CNTF/LIF-mediated transcriptional activation. The CyRE C/EBP-related sites interact with nuclear proteins from the human neuroblastoma cell line, NBFL, including a novel, protein synthesis-dependent, nuclear protein complex, induced by CNTF treatment. These nuclear proteins are not, however, recognized by antisera to known C/EBP proteins. Therefore, other nuclear proteins regulated by independent pathways act in concert with the JAK-STAT pathway to mediate CNTF/LIF regulation of VIP gene expression through the CyRE.

Base Sequence↗
PubMed1995

Quantitative analysis of the expression of a VIP transgene.

We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

Animals↗
PubMed1995

Adenosine antagonists potentiate D2 dopamine-dependent activation of Fos in the striatopallidal pathway.

Adenosine antagonists potentiate dopamine-mediated behaviours. A2a adenosine and D2 dopamine receptors are abundantly co-expressed within the striatopallidal subset of striatal neurons, suggesting that this is the site of interaction between A2a and D2 receptors. We show that the D2-dependent induction of the immediate early gene c-Fos occurs in striatopallidal neurons 3 h following injection of reserpine (10 mg/kg). We used this paradigm to test whether adenosine antagonists modulate D2-dependent activation of striatopallidal neurons. The non-selective A1-A2 adenosine antagonists theophylline (25 mg/kg) or 3,7-dimethyl-1-propargylxanthine (25 mg/kg) potentiated the effect of a submaximal dose of the D2 dopamine agonist quinpirole (0.05 mg/kg) to prevent the induction of striatal c-Fos following reserpine. Co-administration of the A2a receptor antagonist 8-(3-chlorostyryl) caffeine (5 mg/kg) with quinpirole (0.05 mg/kg) also attenuated striatal c-Fos induction following reserpine to a greater extent than 0.05 mg/kg quinpirole alone. When administered prior to reserpine, theophylline (25 mg/kg) or 3,7-dimethyl-1-propargylxanthine (25 mg/kg) partially attenuate the induction of striatal c-fos. These results demonstrate that systemic administration of adenosine antagonists enhance D2 dopamine receptor-dependent regulation of c-Fos in the striatopallidal pathway. These results support a functional interaction between A2a adenosine and D2 dopamine receptors in striatopallidal neurons.

Adenosine↗
PubMed1995

Differences in nuclear signaling by leukemia inhibitory factor and interferon-gamma: the role of STAT proteins in regulating vasoactive intestinal peptide gene expression.

To investigate the importance of STAT protein activation in leukemia inhibitory factor (LIF)-mediated induction of neuropeptide gene transcription, we compared signaling to the 180-bp cytokine response element (CyRE) in the vasoactive intestinal peptide (VIP) promoter by interferon-gamma (IFN-gamma) and LIF. We show that LIF and IFN-gamma activate STAT proteins but only LIF activates VIP gene transcription. Thus STAT activation is not sufficient for VIP transcriptional activation. In a CyRE reporter plasmid, in which the STAT site has been deleted, LIF, but not IFN-gamma, activates transcription, indicating that sequences within the CyRE distinct from the STAT site are important to LIF-mediated transcriptional activation. The CyRE does not mediate transcriptional activation to LIF in a non-VIPergic cell line, suggesting that cell-specific factors exist which are permissive for cytokine-dependent regulation of gene expression. Human and mouse sequences are highly conserved in the region of the CyRE, consistent with the functional importance of multiple regions of the CyRE. These findings show that regions within the CyRE distinct from the STAT site are important to the LIF-dependent regulation of VIP gene expression and enable the CyRE to respond in a cell-specific and cytokine-specific manner.

Animals↗
PubMed1995

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion.

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.

Animals↗
PubMed1994

Hereditary variations in monoamine oxidase as a risk factor for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder caused by loss of dopaminergic neurons in the brainstem. Recent studies suggest that several genes may have a role in determining individual susceptibility to this disease, and the degradative enzyme monoamine oxidase (MAO) has been implicated in the disease process. Wide differences in activity levels for both forms of this enzyme (MAO-A and MAO-B) exist in the human population, and levels of both are genetically determined. Here we have compared the frequency of haplotypes at the MAOA and MAOB loci on the X chromosome in 91 male patients with PD and 129 male controls. Alleles were marked using two restriction fragment length polymorphisms (RFLPs), a (GT)n repeat in the MAOA locus, and a (GT)n repeat in the MAOB locus. One particular haplotype marked by the RFLP's at MAOA was three times more frequent in patients with PD as compared with controls, and the overall distribution of these alleles was significantly different (p = 0.03) between these two groups. Another MAOA haplotype was about threefold more common in controls than in patients with PD (p = 0.005). No associations were observed between individual MAOB alleles and the disease state, but the frequency distribution for all alleles was significantly different in the two populations (p = 0.046). These findings support the idea that the MAO genes may be among the hereditary factors that influence susceptibility of individuals to PD.

Adolescent↗
PubMed1994

Coordinate regulation of choline acetyltransferase, tyrosine hydroxylase, and neuropeptide mRNAs by ciliary neurotrophic factor and leukemia inhibitory factor in cultured sympathetic neurons.

The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine-mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40-50% of superior cervical ganglion neurons in dissociated culture.

Animals↗
PubMed1994

Leukemia inhibitory factor and ciliary neurotrophic factor increase activated Ras in a neuroblastoma cell line and in sympathetic neuron cultures.

The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. We have previously studied the regulation of vasoactive intestinal polypeptide gene expression by LIF and CNTF in the NBFL neuroblastoma cell line. Because these cytokines induce tyrosine phosphorylation that may lead to Ras activation, we explored a possible role for Ras in LIF- and CNTF-induced signal transduction. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. The protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, activated Ras, and vasoactive intestinal polypeptide mRNA in NBFL cells. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Multiple regions within the cytoplasmic domains of the leukemia inhibitory factor receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells.

The receptor for leukemia inhibitory factor (LIFR), in combination with the signal-transducing subunit for interleukin-6-type cytokine receptors, gp130, and LIF, activates transcription of acute-phase plasma protein genes in human and rat hepatoma cells and the vasoactive intestinal peptide gene in a human neuroblastoma cell line. To identify the regions within the cytoplasmic domain of LIFR that initiate signal transduction independently of gp130, we constructed a chimeric receptor by linking the extracellular domain of the granulocyte colony-stimulating factor receptor (G-CSFR) to the transmembrane and cytoplasmic domain of human LIFR. The function of the chimeric receptor protein in transcriptional activation was assessed by G-CSF-mediated stimulation of cotransfected cytokine-responsive reporter gene constructs in hepatoma and neuroblastoma cells. By using the full-length cytoplasmic domain and mutants with progressive carboxy-terminal deletions, internal deletions, or point mutations, we identified the first 150 amino acid residues of LIFR as the minimal region necessary for signaling. The signaling reaction appears to involve a cooperativity between the first 70-amino-acid region containing the two sequence motifs conserved among hematopoietin receptors (box 1 and box 2) and a critical sequence between residues 141 and 150 (box 3). Analogous analyses of the cytoplasmic domains of G-CSFR and gp130 indicated similar arrangements of functional domains in these receptor subunits and the requirement of a box 3-related motif for signaling.

Amino Acid Sequence↗
PubMed1994

STAT proteins participate in the regulation of the vasoactive intestinal peptide gene by the ciliary neurotrophic factor family of cytokines.

Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are members of a family of neuropoietic cytokines that have a broad range of actions on many different neuronal populations. In cultured sympathetic neurons, CNTF and LIF induce transcription of the VIP and other neuropeptide genes as part of a program of differentiation. To gain insight into the nuclear events involved in cytokine-mediated activation of the neuropeptide genes involved in neuronal differentiation, we have investigated the mechanisms of transcriptional activation of the vasoactive intestinal peptide (VIP) gene by the CNTF family of cytokines. In the neuroblastoma cell line NBFL, CNTF, LIF, and a related cytokine, oncostatin-M, activate VIP gene transcription through a 180-base pair cytokine response element (CyRE). Deletion analysis of the VIP CyRE showed that multiple regions within the 180 base-pairs are important for cytokine-mediated transcriptional activation of the VIP gene. To one of these regions within the CyRE, cytokine treatment induces binding of a protein complex composed of members of the signal transducers and activators of transcription (STAT) transcription factor family. Mutation of this STAT-binding site attenuates cytokine-mediated transcriptional activation. LIF treatment of primary sympathetic neurons also induced binding of a STAT-containing protein complex to the VIP CyRE. Thus, activation of STAT transcription factors contributes to the induction of the VIp gene by the CNTF family of cytokines and may be involved in cytokine-mediated differentiation of sympathetic neurons.

Base Sequence↗
PubMed1993

Differential localization of A2a adenosine receptor mRNA with D1 and D2 dopamine receptor mRNA in striatal output pathways following a selective lesion of striatonigral neurons.

We have used the suicide transport agent volkensin to produce selective lesions of striatonigral neurons. By in situ hybridization histochemistry unilateral volkensin injections in the substantia nigra decreased the number of D1 receptor mRNA-expressing neurons in the ipsilateral striatum but did not change the number of D2 receptor and A2a adenosine receptor mRNA-expressing neurons. These findings confirm that striatonigral neurons express D1 receptors and suggest that D2-A2a receptor expressing neurons are predominantly localized to other neuronal populations within the striatum.

Animals↗
PubMed1993

Ciliary neurotrophic factor coordinately activates transcription of neuropeptide genes in a neuroblastoma cell line.

Differentiation factors have been identified that influence the phenotype of sympathetic neurons by altering expression of classical neurotransmitters and neuropeptides. Investigation of the molecular mechanisms through which such factors act would be facilitated by the availability of a neuronal cell line that responds to these factors in a fashion similar to sympathetic neurons. We have identified a human neuroblastoma cell line, NBFL, that responds to the differentiation factor ciliary neurotrophic factor (CNTF) by coordinately inducing multiple neuropeptide genes as do sympathetic neurons. Treatment of NBFL cells with CNTF increases vasoactive intestinal polypeptide (VIP), somatostatin, and calcitonin gene-related peptide (CGRP) mRNAs but does not change other neurotransmitter properties. The induction of VIP mRNA by CNTF in NBFL cells is dose dependent, rapid, sustained, and independent of new protein synthesis. Genomic 5' flanking sequences located within a 1.59-kilobase region of the human VIP gene and distinct from the previously defined cAMP-responsive element subserve transcriptional activation by CNTF. Further examination of NBFL cells should permit the elucidation of the molecular mechanisms by which CNTF and other differentiation factors coordinately activate neuropeptide gene transcription to influence neuronal differentiation. Similar mechanisms may mediate the effect of CNTF on neuronal survival.

Calcitonin Gene-Related Peptide↗
PubMed1993

Calcium regulation of vasoactive intestinal polypeptide mRNA abundance in SH-SY5Y human neuroblastoma cells.

Second messenger regulation of gene expression provides a mechanism by which neurons can transduce environmental stimuli into long-term changes in the expression of molecules involved in neuronal signaling. We have investigated calcium-dependent induction of vasoactive intestinal polypeptide (VIP) mRNA and compared it with induction of VIP mRNA by cyclic AMP. Depolarization with 60 mM KCl or exposure to the calcium ionophore A23187 increases VIP mRNA levels in SH-SY5Y cells. The increase in VIP mRNA content in response to Ca2+ mobilization is slow, independent of adenylate cyclase activation, and requires de novo protein synthesis. The increase in VIP mRNA content in response to elevation of cyclic AMP levels by forskolin/isobutylmethylxanthine is independent of Ca2+ influx and does not require new protein synthesis. The mRNA for the transcription factors ATF-3, c-fos, c-jun, junB, and zif/268 is induced by A23187. Of these, ATF-3 showed the greatest relative induction by A23187 compared with induction by forskolin/isobutylmethylxanthine.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

Amino Acid Sequence↗